Cholinergic Ligand–dependent Modulation of Oxidative Phosphorylation Coupling in Digitonin-permeabilized BE(2)-C Neuroblastoma Cells

Summary

We present a protocol to selectively permeabilize plasma membrane, enabling targeted delivery of substrates and inhibitors to assess mitochondrial oxidative phosphorylation (OXPHOS) in cultured cells, overcoming plasma membrane transport barriers that typically hinder such analysis. Using this technique, we demonstrated how OXPHOS function is modulated by muscarinic agonists and antagonists.

Abstract

The objective of this study was to assess oxidative phosphorylation (OXPHOS) function in cultured cells using defined substrate–inhibitor combinations while retaining cellular structure and cytosolic context lost in isolated mitochondrial preparations. Because intact cells are poorly permeable to several Krebs cycle intermediates, direct assessment of substrate-supported respiration through specific electron transport chain (ETC) entry points is limited. To overcome this, we applied digitonin-mediated selective plasma membrane permeabilization and performed extracellular flux analyzer–based coupling and electron flow assays in BE(2)-C neuroblastoma cells. To determine cell-type dependence, digitonin was empirically titrated in HEK293 cells and primary rat dorsal root ganglion (DRG) neurons using succinate + rotenone to isolate Complex II–IV–driven respiration.

Succinate-supported respiration with Complex I inhibition showed increased Complex II–IV–driven oxygen (O₂) consumption in permeabilized compared with non-permeabilized cells, consistent with improved access of a membrane-impermeant substrate to mitochondria. In contrast, respiration supported by substrates that enter via endogenous transport pathways (e.g., pyruvate/malate) showed smaller differences between conditions. Using this platform to test muscarinic ligands, we observed agonist- versus antagonist-associated differences in O₂ consumption in the coupling assay, whereas the electron flow assay revealed minimal ligand-associated effects under the tested conditions. These findings indicate that detectable ligand effects were more prominent at the level of coupling-defined respiratory states than maximal electron transfer capacity. Overall, selective permeabilization expands substrate accessibility in cultured-cell bioenergetic assays and enables analysis of pharmacologic modulation of mitochondrial respiration.

Introduction

Malfunction or reprogramming of mitochondrial energy metabolism is central to a wide spectrum of disorders, including neurodegeneration, cancer, inflammatory diseases, cardiometabolic conditions, and various genetic disorders. To better understand the role of mitochondrial metabolism in disease pathogenesis, several analytical strategies have been developed. These include measurements of O₂ consumption rate (OCR), extracellular acidification rate (ECAR), ATP quantification, substrate utilization, profiling or Krebs cycle metabolite analysis, and isotope tracing¹. Among these techniques, OCR serves as a key indicator of mitochondrial respiration and can be assessed using instruments such as High-Resolution Respirometry (HRR; e.g., Oroboros O₂k)², Clark-type electrode, or the extracellular flux analyzer

The Oroboros O₂k system measures O₂ concentration in a sealed chamber using Clark type polarographic O₂ sensor (POS)^3,4,5 and is suitable for samples such as isolated mitochondria, permeabilized cells, and tissue homogenates. It consists of a gold/platinum cathode, a silver anode, and a KCl electrolyte reservoir separated from the sample by a 25 µm O₂ permeable and chemically resistant Fluorinated Ethylene Propylene membrane (FEP). The Clark-type electrode³ also operates in a closed chamber, detecting changes in O₂ concentration via an O₂-sensitive electrode, and is commonly used for isolated mitochondria or cell suspensions. In contrast, the extracellular flux analyzer quantifies OCR and ECAR in live adherent cells or isolated mitochondria using fluorescent sensors embedded in microplates, enabling high-throughput metabolic profiling. A practical advantage of the extracellular flux analyzer platform is automated, multiparametric assessment across multiple conditions in parallel, whereas closed-chamber systems (e.g., HRR or Clark-type electrodes) typically provide lower throughput and require more manual handling.

The extracellular flux analyzer is especially useful for evaluating mitochondrial function in intact adherent cells or suspension cells that settle at the bottom of the microplate. In assays using intact adherent cells, mitochondrial energy metabolism can be profiled using substrates such as glucose or pyruvate⁶. Alternatively, assays using small quantities of enriched mitochondrial fractions allow sequential measurement of basal respiration, ADP-stimulated respiration (State 3), resting respiration (State 4), and uncoupler-stimulated respiration using combinations of substrates and inhibitors⁷. However, in intact cell systems, Tricarboxylic acid (TCA) cycle metabolites that are not cell-permeable cannot be used to assess their metabolism directly. While isolated mitochondria allow the use of such metabolites due to the absence of plasma membrane barriers. Analysis of isolated enriched mitochondrial bioenergetics approach has limitations: mitochondrial structure can be altered during isolation, and cytosolic factors that influence mitochondrial metabolism and metabolite transport are reduced or absent. Accordingly, selective permeabilization approaches are often used as a compromise that can retain aspects of cellular architecture while enabling controlled access to mitochondria. To overcome the limitations of using either intact cells or isolated mitochondria for metabolic assays, this protocol describes a method for permeabilizing adherent neuroblastoma (BE(2)‑C) cells using digitonin. This approach facilitates import of otherwise non–cell-permeant metabolites across the plasma membrane while maintaining functional mitochondrial responses to defined substrates and inhibitors.

The selectivity of digitonin is based on differences in membrane lipid composition: the plasma membrane is rich in cholesterol, whereas mitochondrial membranes—particularly the inner mitochondrial membrane—contain very little cholesterol⁸. At low concentrations, digitonin (or saponin) selectively and completely permeabilizes the plasma membrane due to its high cholesterol content, while mitochondrial membranes are affected only at higher concentrations^9,10. Digitonin binds to cholesterol and forms pores in the plasma membrane, allowing small molecules such as TCA cycle intermediates to enter the cytosol without disrupting mitochondrial structure or function when properly optimized. This approach is best suited for cultured cells that can be plated reproducibly as adherent monolayers in an extracellular flux analyzer microplate and requires empirical titration of digitonin concentration for each cell type and seeding density. Operational indicators of preserved mitochondrial integrity include robust, inhibitor‑sensitive OCR responses (e.g., expected decreases with rotenone/antimycin A and intact Complex IV‑driven respiration with ascorbate/TMPD), along with the absence of OCR collapse that would signal overpermeabilization. Therefore, digitonin optimization should be performed before experimental comparisons and repeated whenever culture conditions or cell density change. Moreover, the optimal digitonin concentration varies by cell type and must be empirically determined to establish a permeabilization window that permits entry of impermeant substrates (e.g., succinate) while maintaining inhibitor‑sensitive mitochondrial function. In this study, we illustrate this principle by optimizing digitonin not only in BE(2)-C cells, but also in HEK293 cells and primary rat dorsal root ganglion (DRG) neurons.

This protocol was demonstrated using BE(2)-C neuroblastoma cells, which were originally isolated from the brain of a male patient with neuroblastoma. BE(2)-C cells express various muscarinic receptors, particularly the cholinergic receptor muscarinic type-1 (CHRM1). In our previous studies, we demonstrated that CHRM1 is associated with mitochondria in both the cell bodies and neurites of cultured rat dorsal root ganglion neurons, as well as in CHRM1‑transfected HEK293 cells¹¹. Furthermore, genetic deletion of Chrm1 in mice alters multiple aspects of mitochondrial structure and function^12,13. Therefore, we hypothesize that BE(2)-C cells can be used to investigate the modulatory effects of muscarinic ligands on OXPHOS function. In this demonstration, we examined the effects of muscarinic agents—including agonist acetylcholine, the biased antagonist pirenzepine¹⁴, and the antagonist atropine—on mitochondrial respiration in the presence of either succinate or pyruvate/malate as substrates and compared their impact.

Protocol

1. Thawing and initial plating

NOTE: The base medium for BE(2)-C cells is a 1:1 mixture of ATCC-formulated Eagle's Minimum Essential Medium and F12 Medium, supplemented with 10% fetal bovine serum (FBS).

1. Thaw frozen BE(2)-C cells from a cryovial in a 37 °C water bath. Once thawed, centrifuge the cells 200 × g for 5 min to remove the cryoprotectant.
2. Resuspend the cell pellet in complete growth medium and plate the cells in a 10 cm cell culture dish. Incubate the culture at 37 °C in a humidified incubator with 5% CO₂ in air until the cells reach sub-confluency.

2. Subculturing

1. Subculture the cells for 2–3 passages to ensure they reach a stable exponential growth phase.

3. Trypsinization and single-cell suspension preparation

1. When the culture reaches 60–80% confluency, trypsinize the cells. Pass the cell suspension several times through fine-tip transfer pipettes with polished tips to dissociate clumps and achieve a single-cell suspension.
2. Add fresh medium containing FBS to neutralize the trypsin. Centrifuge the cells at 200 × g for 5 min to pellet them.

4. Cell seeding protocol for 24-well cell culture microplates used in extracellular flux analyzer

1. Resuspend the pellet in complete medium at a final concentration of 10,000 cells per 100 µL per well.
2. Seed the cells at an optimized density to ensure they reach full confluency within 1–2 days. Leave either the four corner wells or a single corner well empty, depending on the experimental design. These wells will serve as background controls.
   NOTE: This may require trial and error, including adjustments to media volume and timing.
3. Monitor the culture for changes in media color—yellowing indicates a drop in pH and necessitates immediate replacement with fresh medium to maintain cell health.

5. Preparation for extracellular flux analyzer-based assay

NOTE: The coupling and electron flow assay formats used here follow established microplate-based mitochondrial respiration workflows⁷, with modifications including permeabilization of BE(2)-C cells using digitonin titration and muscarinic ligand treatment conditions.

1. Prepare stock solutions and 2x Mitochondrial Assay Solution (MAS). Prepare 2x MAS mixes and working reagent (substrates and inhibitors) stock solutions (see Table 1, Table 2, and Table 3) in advance.
2. Store all stock solutions and MAS at -20 °C for future use, allowing for efficient assay setup.
   ​SAFETY: Rotenone, antimycin A, oligomycin, FCCP, digitonin, and TMPD are hazardous and should be handled in accordance with institutional safety guidelines. All inhibitor stock solutions should be prepared and handled in a certified chemical fume hood while wearing appropriate personal protective equipment (lab coat, nitrile gloves, and eye protection). Solutions containing these reagents, as well as DMSO-containing waste, should be collected and disposed of as hazardous chemical waste according to institutional and local regulations. Work surfaces should be decontaminated after use.
3. Hydrating the sensor cartridge
   1. The night before the experiment, hydrate the extracellular flux analyzer sensor cartridge by adding 1 mL of sensor cartridge calibration buffer or phosphate-buffered saline (PBS, pH 7.4) to each well. Let the sensor cartridge sit overnight (12–18 h) at room temperature (20–25 °C).
      ​NOTE: Incubation in a non-CO₂ incubator at 37 °C is not necessary, as the cartridge will equilibrate to room temperature during the loading process. Once placed in the extracellular flux analyzer for calibration, the cartridge will reach 37 °C within approximately 15 min.

6. Day of the experiment

NOTE: Prepare all solutions fresh, adjust pH if necessary, and keep on ice until use.

1. Thaw frozen substrate stocks and 2x MAS at room temperature (20–25 °C) for 15–30 min or in a 37 °C water bath for 5–10 min.
2. Prepare all substrate and injection solutions (see Table 4, Table 5, Table 6, and Table 7) and verify that the pH is at 7.4. Adjust if necessary and keep the solutions on ice until use. Use Table 4 and Table 5 for the coupling assay. For electron flow assay, use Table 6 and Table 7.
3. Load the inhibitors and substrates into the appropriate sensor cartridge ports and prepare to run the assay. Depending on the type of assay—coupling assay or electron flow assay ⁷ —refer to Table 4 and Table 5 or Table 6 and Table 7, respectively, as needed.
4. Prepare all substrate and inhibitor working solutions to defined final volumes (bulk mixes: 15 mL; cartridge injection mixes: 2 mL per port). Calculate concentrations to achieve the desired final in-well concentrations after injection (final well volume: 700 µL after Port D).
5. Add 450 µL of the prepared assay solution to each well of the 24-well extracellular flux analyzer plate containing muscarinic ligands (final ligand concentration: 1 µM; total well volume: 500 µL). Ensure each well already contains 50 µL of 1x MAS buffer and submerged cells.
   NOTE: Perform this step immediately before loading the plate into the analyzer.
6. Load 50 µL of each prepared reagent into the corresponding injection port of the sensor cartridge.

7. Instrument programming and Assay execution

1. Turn on the extracellular flux analyzer at least 1–2 h before the experiment to allow the internal incubator to stabilize at 37 °C. Maintain the chamber at 37 °C throughout calibration and the assay run.
2. Program the analyzer to perform the appropriate mix, wait, and measurement cycles according to Table 8.
3. Label wells and experimental groups in the software according to the experimental design.
4. Start the assay run. Allow the instrument to calibrate the sensor cartridge (~15 min).
5. Prepare the assay plate for loading during cartridge calibration. Delay loading if necessary; the sensor cartridge will remain stable at 37 °C inside the instrument.
6. Complete the assay when the final measurement cycle after Port D injection finishes. Proceed immediately to post-assay normalization and data analysis.

8. Cell plate preparation for extracellular flux analyzer assay

1. Begin preparing the cell plate immediately after placing the sensor cartridge into the analyzer for calibration. Add 700 µL of 1x MAS buffer to each well containing adherent cells. Incubate at room temperature (20–25 °C) for 1 min.
2. Carefully aspirate the buffer without disturbing attached cells. Repeat the wash step for at least 3 x 1 min to remove residual culture medium and complete all washes within 10 min.
3. Add 50 µL of 1x MAS buffer to a designated background well (no cells) to establish a visual volume reference.
4. Aspirate buffer from experimental wells, leaving approximately 50 µL of residual buffer in each well.
5. Incubate the plate at 37 °C in a water bath, keeping the plate above the water surface to prevent evaporation. Maintain at 37 °C for 10 min (maximum 20 min) before loading.
6. Immediately before loading the plate, add 450 µL of 1x MAS buffer containing the appropriate substrates to each well to reach a final volume of 500 µL.
7. Insert the plate into the extracellular flux analyzer.

9. End of assay and data analysis

1. Remove the cell plate and sensor cartridge from the analyzer after completion of the run.
2. Normalize OCR values per well using total protein quantification or total cell counts. Lyse cells with RIPA buffer and quantify total protein using the Bradford assay or another spectrophotometric protein assay.
3. Normalize OCR values using the analysis software and export data as needed.
4. Apply the following acceptance criteria: confirm baseline OCR stability (≤15% drift across two consecutive measurement cycles) and confirm ≥60% OCR reduction following rotenone, oligomycin, or antimycin A injection.
5. Exclude runs that fail these criteria and re-optimize permeabilizer concentration or cell seeding density as needed.

10. Exporting results

1. Insert a USB drive into the instrument.
2. Export the assay data.

11. Data processing

1. Analyze data using the instrument software.
2. Export processed data in formats compatible with analysis software of choice for further analysis and visualization.

Results

Optimization of digitonin concentration for BE(2) C Cell permeabilization in coupling assay
At the beginning of this protocol, we standardized the optimal digitonin concentration required for effective cell permeabilization without compromising mitochondrial respiration. In this protocol, we tested 5 µM and 10 µM digitonin as starting concentrations and performed a coupling assay. This assay evaluates the degree of coupling between the ETC and the OXPHOS machinery, allowing us to distinguish between ...

Discussion

This study introduces a rapid and cost-effective protocol for evaluating muscarinic ligand-mediated modulation of OXPHOS in cultured neuroblastoma cells. By employing the selective plasma membrane permeabilizer digitonin, in combination with defined substrate-inhibitor pairs, we enabled targeted delivery of metabolic substrates such as succinate directly to mitochondria in situ. This approach overcomes the limitations of intact cell membranes, which typically restrict access to key TCA cycle intermediates. Impor...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acetylcholine chloride	MilliporeSigma	A6625	Muscarinic agonist
ADP	MP Biomedicals, Inc	ICN19014901	Electron acceptor, substrate for ATP synthesis.
Antimycin A	AAJ63522MA	Thermo Scientific	Inhibits mitochondrial complex III
Atropine	MilliporeSigma	1044990	Muscarinic antagonist
Be2C cells	American Type Culture Collection	CRL-2268	A neuroblast cell that was isolated from the brain of a male patient with neuroblastoma, express muscarinic receptor
Digitonin	MilliporeSigma	D141	Mild detergent, membrane permeabilizer.
D-Mannitol	Thermo Scientific Chemicals	AA3334236	A non-metabolizable sugar alcohol, used for osmotic stabilization and membrane protection
EGTA	MilliporeSigma	E0396	Calcium Chelation, Prevents activation of calcium-dependent proteases and other enzymes that could degrade mitochondrial proteins.
fatty acid-free BSA	MilliporeSigma	126575	Prevents uncontrolled fatty acid effects, stabilizes mitochondria
FCCP	Cayman Chemical	NC0904863	A protonophore and uncoupler of oxidative phosphorylation in mitochondria
HEPES	MilliporeSigma	391340	pH buffering, compatibility with mitochondrial function
L-Ascorbate	TCI America	A053925G	TCA cycle intermediate, substrate of OXPHOS
Magnesium chloride	MilliporeSigma	M8266	For ionic balance, support for enzymatic activity
N1,N1,N1,N1-tetramethyl-1,4-phenylene diamine (TMPD)			Electron Donor to Cytochrome c, assessment of complex IV function by bypassing Complex I and II, TMPD/ascorbate enables direct measurement of Complex IV-dependent oxygen consumption
Oligomycin	MilliporeSigma	1404-19-9	Inhibits mitochondrial ATP synthesis, Complex V
Pirenzepine dihydrochloride	MilliporeSigma	P7412	Muscarinic biased agonist
Pyruvic acid	Thermo Scientific Chemicals	AC132155000	TCA cycle substrate
Rotenone	MilliporeSigma	557368	Inhibits mitochondrial complex I
Seahorse XF24 V7 PS Cell Culture Microplates	Agilent	102340-100	Cell Culture Microplates
Seahorse XFe24 Analyzer	Agilent	S7801BR	Agilent Seahorse XFe24 Analyzers measure the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of live cells in a 24-well plate format.
Succinic acid	Fisher Chemical	AC158742500	TCA cycle intermediate, substrate of OXPHOS
XFe24 sensor cartridge calibration buffer	Agilent	102340100	PBS buffer pH 7.2
XFe24 sensor cartridges	Agilent	102340-100	sensor for OCR and ECAR measurement